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October - December 2007: 
Volume 20, Issue 4

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ARCHIVE

Pulmonary Surfactant
Abstract
SUMMARY. Pulmonary surfactant is a complex mixture of lipids and proteins that constitutes the mobile liquid phase covering the large surface area of the alveolar epithelium. It maintains minimal surface tension within the lungs in order to avoid lung collapse during respiration. The components of surfactant are synthesised within the lung in the alveolar type II cells, Clara cells and submucosal cells. Surfactant components are also produced in small quantities at non-pulmonary sites (e.g. trachea, brain, testes, salivary glands, lachrymal glands, heart, prostate, kidney, pancreas). Pulmonary surfactant is composed of phospholipids, neutral lipids and surfactant proteins (SP). The SPs are divided into the hydrophobic SP-B and SP-C and the hydrophilic SP-A and SP-D. SP-B and SP-C lower the alveolar surface tension, while SP-A and SP-D are primarily concerned with host-defense functions, acting as immune mediators. They may aggregate or agglutinate pathogens, recruit and activate neutrophils and macrophages, induce phagocytosis, offer resistance to allergen challenge by interfering with allergen–IgE interaction, mast cell/basophil degranulation, cellular infiltration and helper Tcell polarization, and they are involved in the clearance of apoptotic and necrotic cells. Pulmonary surfactant abnormalities have been implicated in various human diseases, such as obstructive lung disease (asthma, bronchiolitis, chronic obstructive pulmonary disease [COPD]), infectious and suppurative lung diseases (cystic fibrosis [CF], pneumonia, and human immune deficiency virus [HIV]), adult respiratory distress syndrome (ARDS), pulmonary oedema, chronic lung disease of prematurity and SP-B deficiency, interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis [IPF], hypersensitivity pneumonitis), pulmonary alveolar proteinosis, and following cardiopulmonary bypass and lung transplantation, and in smokers. Pneumon 2007; 20(4):364–371
Full text
Introduction

The lung is exposed continuously to inhaled pathogens, pollutants and particles. Therefore, the pulmonary immune system needs to provide defence against harmful pathogens and to prevent inappropriate inflammatory response. Several defence mechanisms contribute to the innate immunity of the lung, including filtration in the naso-oropharynx, sneezing, coughing, mucociliary clearance, opsonins (Ig), innate immune cells (AM, Neu), and surfactant proteins (SP)1.

Pulmonary SPs comprise a complex mixture of lipids and proteins that forms the mobile liquid phase covering the large surface area of the alveolar epithelium. It maintains minimal surface tension within the lungs in order to avoid lung collapse during respiration2. Recent studies have shown that SPs also function in the pulmonary host defence as immune mediators3,4.

PULMONARY SURFACTANT LIPIDS

Lung surfactant consists of a unique and complex mixture of lipids (85-90%) and specific proteins (10%). Surfactant lipids are mostly phospholipids (90%) that are essential for reducing surface tension within the lungs, with 10% of neutral lipids, such as triglycerides and cholesterol4.

The most abundant surfactant phospholipid is phosphatidylcholine, mostly in the form of desaturated dipalmitoylphosphatidylcholine [DPPC], which plays an essential role in decreasing surface tension2,3. The hydrophilic component of DPPC is oriented towards the liquid surface of the alveolar air-water interface, while the hydrophobic component, palmitic acid, is oriented towards the air phase4.

Pulmonary Surfactant Proteins (SP)

About 10% of surfactant consists of proteins. Four SPs have been identified: SP-A, SP-B, SP-C, and SP-D. SPs are divided into the hydrophobic surfactant proteins, SP-B and SP-C, and the hydrophilic surfactant proteins, SP-A and SP-D. While SP-B and SP-C lower the alveolar surface tension, SP-A and SP-D primarily mediate the host-defence function of pulmonary surfactant3. The biophysical and immunological functions of pulmonary surfactant are listed in Table 13.



Surfactant Synthesis and Secretion

Lung surfactant is synthesized in the alveolar type II cell. Its lipids and hydrophobic proteins (SP-B and SP-C) are stored in lamellar bodies and secreted by regulated exocytosis. It is then secreted into the alveolar space to form a lattice called ‘tubular myelin’, which is considered to be an intermediate product that forms the monolayer lipid film covering the alveolar space. In contrast, the hydrophilic proteins (SP-A and SP-D) appear to be secreted independently of lamellar bodies4.

Although the main site of surfactant synthesis is the lung, SP protein mRNA has been found in non-pulmonary sites, including the trachea, brain, testes, salivary glands, lachrymal glands, heart, prostate, kidney, pancreas and the female urogenital tract. However, it is not yet clear whether all these organs express sufficient amounts of SP to be physiologically effective1.

Alveolar type II cells synthesize all four of the SPs and surfactant lipids, which are packaged together in the lamellar body, which is a unique secretory organelle. Following appropriate stimulation, such as birth or a deep breath5,6, the lamellar body contents are secreted into the thin liquid hypophase that covers the alveolar epithelium6,7, (Figure 1). In a process facilitated by SP-B, a monolayer film, enriched with phosphatidylcholine covers the alveolar space, maintaining minimal surface tension within the lungs in order to avoid lung collapse during respiration4.

SP-A, SP-B and SP-D are also synthesized by airway cells, including Clara cells and submucosal cells. It is not known whether the functions of the SPs secreted by airway cells are similar to those of the SPs secreted by alveolar cells, but the lack of lipid in airway secretory products indicates that these products do not participate in surface tension reduction4.

Figure 1. Surfactant is synthesized by alveolar type II cells and stored as intracellular inclusion organelles called ‘lamellar bodies’. It is then secreted into the alveolar space to form a lattice called ‘tubular myelin’, which is considered to be an intermediate product that forms the monolayer lipid film covering the alveolar space4. Following appropriate stimulation, such as birth or a deep breath5,6 lamellar-body contents are secreted into the thin liquid hypophase that covers the alveolar epithelium6,7.


Surfactant and Lung host-defense mechanisms

Recent studies have provided compelling support for the role of SP-A and SP-D as mediators of various immunecell functions1. They defend against pulmonary pathogens through their ability to aggregate or agglutinate the pathogens, and to recruit and activate neutrophils and macrophages, thereby inducing phagocytosis, and they have bacteriostatic and fungistatic effects on microbial growth. SP-A and SP-D also offer resistance to allergen challenge by interfering with allergen–IgE interaction, mast cell/basophil degranulation, cellular infiltration and helper T-cell polarization. They are also involved in the clearance of apoptotic and necrotic cells1,8.

Figure 2. Collectins SP-A and SP-D structure (modified from ref. 1).


SP-A and SP-D are members of the collectin family of proteins. Collectins are distinguished by their amino (N)- terminal collagen-like regions that have a repeating triple helix of Gly-X-Y triplets. The carboxy-terminal domains of the collectins all have C-type (calcium-dependent) lectin activity1,4. The collectins are assembled as trimeric subunits, which multimerize to varying degrees. SP-A is an octadecamer and forms a bouquet-like structure (Figure 2), whereas SP-D forms a dodecamer. The lectin domains mediate the interaction of collectins with a wide variety of pathogens (Figure 3). The consequence of this interaction is pathogen opsonization and enhanced uptake by phagocytes (Figure 3)1,8.

Figure 3. Collectins SP-A and SP-D bind to a variety of bacteria, viruses, allergens and apoptotic cells, functioning as opsonins to enhance the uptake and phagocytosis of these cells and particles (modified from Wright, 20051).

SP-D is also a collagen-like glycoprotein with structural and biophysical properties similar to those of SP-A, although it is even more soluble in the aqueous alveolar environment4,8. Table 2 summarizes the immunological lung diseases (asthma, bronchiolitis, chronic obstructive pulmonary disease [COPD], and following lung transplantation), infectious and suppurative lung diseases (cystic fibrosis [CF], pneumonia, and human immune deficiency virus [HIV]), adult respiratory distress syndrome (ARDS), pulmonary oedema, other diseases specific to infants (chronic lung disease of prematurity, and SP-B deficiency), interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis [IPF], and hypersensitivity pneumonitis) and pulmonary alveolar proteinosis (PAP), and following cardiopulmonary bypass, and in smokers3,9.



Asthma

Clinical studies have suggested that surfactant from asthmatics is functionally impaired, possibly due to the influx of inhibitory proteins into the airways, although changes in surfactant composition may occur9,10. In chronic or acute asthma, products of inflammatory cells (including proteases and reactive oxygen and nitrogen species) and airway oedema may contribute to surfactant dysfunction9. At present, the contribution of surfactant in the asthmatic process is unclear.

Animal models of asthma suggest either decreased amounts of surfactant secreted or increased uptake of the extracellular surfactant.

The clinical use of surfactant therapy in asthma is currently under investigation. In a study of 12 asthmatic children given aerosolized surfactant, there was no change in FVC, FEV1, or PEF9. However, in another trial, where 11 adult asthmatic patients were given aerosolized surfactant after an asthma attack, all patients showed improvement in pulmonary function9. Larger trials are needed to evaluate these findings.

Smoking and Chronic Obstructive Pulmonary Disease (COPD)

Smoking might affect surfactant homeostasis altering surfactant composition and function. Smokers are likely to have a decrease in the phospholipid content and impairment of the surface activity of surfactant recovered from bronchoalveolar lavage (BAL)10. Wirtz and Schmidt11 showed that type II pneumocytes exposed directly to cigarette smoke in culture have decreased secretion of phosphatidylcholine11.

The cumulative effects of cigarette smoking on lung surfactant, due to airway inflammation and the subsequent products of activated neutrophils and macrophages, are likely to result in a significant impairment of surface tension reduction across the alveolar wall. The increased surface tension in the chronic smoker may contribute to alveolar wall rupture and the development of emphysema9.

Host defence functions of surfactant may also be impaired in the chronic smoker12. Levels of both SP-A and SP-D are decreased in BAL fluid from chronic smokers, which may contribute to their increased incidence of respiratory infections12. In a study by Betsuyaku et al13 SP-A was observed to decrease with age alone or due to the cumulative effects of smoking and the development of emphysema, while SP-D decreased due to long-term smoking13.

There is still limited information on the value of surfactant treatment in COPD patients. In a single study on the effect of surfactant phospholipid in COPD, patients who received aerosolized treatment for two weeks had a modest improvement in lung function tests compared to those who received placebo9.

Cystic Fibrosis (CF)

Studies on BAL fluid from patients with CF showed a decrease in the content of intact SP-A and evidence of proteolytic cleavage of SP-A9. The decreased levels of SP-A may predispose to lung infection in these patients. The surface tension reduction of surfactant is also impaired in CF patients, and alterations in surfactant lipid composition may contribute to this impairment9.

Pneumonia

Alterations in PS composition have been encountered in patients with pneumonia. Surfactant in BAL fluid from these patients has qualitative alterations in lipid content, similar to those observed in patients with ARDS. The amounts of SP-A and SP-D were also decreased and surfactant surface tension reduction function was impaired9,14. Limited experience with selective bronchial instillation of surfactant in patients with pneumonia has suggested the possibility of benefit9,14.

In a recent study by Giannoni et al15 in infected mice deficient in SP-A, SP-D or both collectins (SP-AD-/-), on intratracheal administration of P. aeruginosa, phagocytosis of the bacteria by alveolar macrophages was decreased in SP-A-/- and SP-D-/- mice and there was greater infiltration by neutrophils in the lungs of the deficient mice than in the wild type. The authors concluded that collectins SP-A and SP-D enhance the pulmonary clearance of P. aeruginosa by stimulating phagocytosis by alveolar macrophages, and by modulating the inflammatory response in the lungs15.

Idiopathic Pulmonary Fibrosis (IPF)

Studies in IPF suggest that the total amount of surfactant phospholipids is decreased and their composition is altered. The concentration of surfactant aggregates is also decreased and the surface tension reduction is impaired9.

Increased levels of SP-A in the BAL fluid of IPF patients over normal values have been reported. Furthermore, immunohistochemistry studies showed extensive type II cell hyperplasia, containing greatly increased levels of SP-A. These findings have been confirmed by protein immunoblotting and Western blotting studies in IPF patients16,17.

Recent data suggest that the BAL fluid levels of phospholipid and SP-A, and the SP-A/PL ratio could predict outcome in IPF patients. Takahashi et al18 reported that patients who had a lower SP-A/PL ratio had a five-year survival rate of 30% whereas those who had a greater ratio had a five-year survival rate of 68%18.

The possible benefit of surfactant therapy in IPF has not been yet investigated9, 16-18.

Sarcoidosis

According to most reports, the amount and fractional content of surfactant phospholipid recovered in BAL fluid from patients with sarcoidosis are no different from controls. The surface activity of surfactant is impaired and there is a reduction in the large aggregate pool size17. The mechanisms responsible and the pathophysiologic relevance of these observations are unclear3,9.

Hypersensitivity Pneumonitis

The fractional content of large surfactant aggregates and the phospholipid content of BAL fluid from patients with hypersensitivity pneumonitis is not significantly different those in healthy patients, although slight differences have been reported9. Reports of changes in SP-A levels were conflicting; different studies have reported both significant decreases and increases. SP-B levels were reported to be the same as in controls9,17.

Pulmonary Alveolar Proteinosis (PAP)

PAP is a rare idiopathic disease, characterized by massive accumulation of surfactant in the alveoli. The exact defect is unclear, but it may be related to GM-CSF deficiency or GM-CSF receptor, which contribute to reduction in the clearance of surfactant from the alveoli9,19. There is marked heterogeneity of mass and charge in the SP-A isoforms, and elevation in the content of SP-A, SPB, and SP-C. The traditional method of treating patients with PAP is whole lung lavage with saline. Recent trials, however, have examined the value of recombinant GMCSF administration9,19.

Surfactant Genetics

The human SP-A locus is located on chromosome 10 and consists of two functional genes in opposite transcriptional orientation20. The human SP-D is linked to the SP-A locus and is located proximal to the centromere at about 80–100 kb from the SP-A2 gene20. A number of alleles have been characterized for each SP-A gene. Functional differences between SP-A1 and SP-A2 alleles, and possibly among alleles of each gene have been demonstrated with in vitro expressed human SP-A alleles20. Several polymorphisms have also been identified for SP-D20,21. The human SP-B locus is located on chromosome 2p12-p11.2. A number of polymorphisms have been characterized for SP-B and some of these have been associated with diseases21. In addition, a number of microsatellite markers flanking the SP-B locus have been characterized21. The SP marker alleles as additional parameters may facilitate the study of mechanisms involved in the pathogenesis of, e.g., specific COPD subgroups22, which may in turn help in the understanding of why, for example, only a fraction of smokers develop the disease23,24.

In addition, studies from the authors’ laboratory have detected microsatellite DNA instability (MSI) in sputum cells of patients with COPD, in D29802 marker (10q22). This marker is located next to the SP-A gene, and it exhibited MSI only in COPD patients, and not in non-COPD smokers or asthmatics25. According to these results, the authors assumed that the SP-A protein expression could be alternated in COPD patients and further related to COPD pathogenesis. Currently, a new study is in progress on the expression levels of SP-A protein in lung biopsies of COPD patients in comparison with non-COPD smokers and healthy non-smokers26

Conclusions

For more than 70 years, surfactant was believed to be a soap-like substance that reduced surface tension in the lung and made breathing easier. With the advent of molecular techniques, the collectins SP-A and SP-D were identified as mediators of various immune-cell functions. Recent studies have shown novel roles for these proteins in the clearance of apoptotic cells and the direct killing of microorganisms. Although surfactant replacement therapies have been successful in reducing mortality due to surfactant deficiencies, the possibility that the collectins might be an effective therapy for treating inflammatory or infectious lung disease has not been investigated in patients.

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